Functional expression of, and assay for, functional cellular receptors in vivo

ABSTRACT

Methods and materials for expressing and assaying functional neuronal receptors in neuronal cells, including methods for detecting particular odorant ligand specificity for particular odorant receptors and methods of using such. For example, methods and materials are provided for assaying for functional odor receptors in intact nasal epithelium of mammals such as rats and mice and for using such.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States ProvisionalPatent Application No. 60/045,961 filed May 7, 1997 by Stuart J.Firestein and Haiqing Zhao, and entitled Process For Delivering,Expressing, And Assaying Neuronal Receptors In Neuronal Cells, which ishereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This research may have been partially funded by a United Statesfederal grant from the National Institutes of Health, National Instituteon Deafness, and Other Communicative Disorders. The United StatesGovernment may, therefore, have certain rights to this invention.

BACKGROUND OF THE INVENTION

[0003] Olfactory transduction begins with the binding of an odorantligand to a protein receptor on the olfactory neuron cell surface, thusinitiating a cascade of enzymatic reactions that results in theproduction of a second messenger and the eventual depolarization of thecell membrane (1,2). This relatively straightforward and commonsignalling motif is complicated by the fact that there are severalthousand odorants, mostly low molecular weight organic molecules, andnearly one thousand different receptors (3,4). The receptors are membersof the superfamily of membrane receptors characterized structurally bypossessing seven transmembrane spanning helices, and functionally bybeing coupled to GTP-binding proteins. Other members of this superfamilyrecognize diverse ligands from peptides to biogenic amineneurotransmitters, hormones, drugs, and other organic compounds. Theodorant receptor sub-family is the largest sub-family of G-proteincoupled receptors (GPCRs) but remains in some ways the most enigmaticsince no particular receptor has been definitively paired with anyligand. Strictly speaking vertebrate odorant receptors are classified as“orphan” receptors—receptors with no identified ligand (5,6).

[0004] This situation is especially problematic for understanding codingin the olfactory system and appreciating the nature of the neural imagepassed to the brain by the peripheral transducing cells (7). Currentmodels of olfactory processing have been driven largely by genetic databased on the now well described patterns of receptor gene expression(8,9). Receptors can be grouped into sub-families based on sequencesimilarities, and subfamilies of receptors are known to be expressedwithin one of four restricted topographic zones in the nasal epithelium,although within these general zones expression patterns appear to berandom (10,11). Further, all neurons expressing a particular receptorgene converge to a restricted target in the olfactory bulb (12, 13).However, olfactory neurons typically generate physiological responses tomultiple odorants (14, 15) and if, as most evidence indicates, each cellexpresses only one type of receptor (11, 16), then the receptors must beable to bind a variety of molecules. Thus, while it may be attractive tohypothesize that the genetic categorization of receptor sequencesreflects systematic differences in ligand specificities, and thatgenetic expression patterns underlie a spatial map for odorantsensitivity, experimental validation of these ideas requires knowing thecorrelation between receptor gene sequence and the encoded receptorprotein's binding specificities, i.e. its receptive field.

[0005] Further progress in this area has been limited by the absence ofa reliable and efficient system for expressing and assaying clonedodorant receptors. There appear to be two main obstacles to obtainingodorant receptor expression in a heterologous system. For one, expressedreceptors must be properly targeted to, and inserted in, the plasmamembrane, a process that may require specialized cellular machinery notavailable in heterologous cell culture expression systems. Secondly,even properly inserted receptors must couple to a second messengersystem in order to produce a response that can be assayed (17).Olfactory specific isoforms of second messenger enzymes have beenidentified in olfactory neurons (2), raising the possibility thatreceptor-effector coupling may be highly specific, and that endogenousG-proteins in heterologous cell systems may be unable to produce apowerful enough response to be measured reliably.

[0006] In order to circumvent these two potential difficulties we haveadopted an alternative strategy for odorant receptor expression. On theassumption that olfactory neurons themselves would be the most capablecells for expressing, targeting and coupling odorant receptors, we usedthe nasal epithelium as an expression system, driving expression of aparticular receptor by including it in a recombinant adenovirus (Adv)and infecting rat nasal epithelia in vivo. Adenovirus vectors have beendeveloped as a tool for efficient gene transfer in mammalian cells (18)and have shown promise in a variety of experimental and clinicalapplications (19-23). Here we show that this system effectivelyexpresses a foreign odorant receptor gene that can be convenientlyassayed for specific ligand activation by physiological methods.Additionally we have been able to identify a set of ligands thatactivate a particular receptor. This invention provides conclusiveevidence that the putative odorant receptor genes cloned several yearsago do indeed encode odorant receptors and, for the first time in avertebrate, pairs a particular receptor of known amino acid sequencewith a specific set of odorant ligands.

[0007] List of Definitions

[0008] The following definitions are provided for illustrative purposesonly and are in no way to be construed as narrowing the scope of theinstant invention in any way.

[0009] Depolarize—

[0010] a change in the cell membrane potential to a more positivevoltage.

[0011] Action potential—

[0012] a rapid transient depolarization of the cell membrane lasting 1-5milliseconds.

[0013] Agonist—

[0014] a molecule or substance that can activate a receptor protein orenzyme.

[0015] Antagonist—

[0016] a molecule that binds to or otherwise interacts with a receptorto inhibit the activation of that receptor or enzyme.

[0017] Ligand—

[0018] a naturally occurring or synthetic compound that binds to aprotein receptor.

[0019] Odorant ligand—

[0020] a ligand compound that, upon biding to a receptor, leads to theperception of an odor including a synthetic compound and/orrecombinantly produced compound including agonist and antagonistmolecules.

[0021] Odorant receptor—

[0022] a receptor protein normally found on the surface of olfactoryneurons which, when activated (normally by binding an odorant ligand)leads to the perception of an odor.

[0023] Olfactory receptor—

[0024] refers to the primary sensory neuron in the nasal epitheliumwhich responds to odors or other ligands.

[0025] Receptor—

[0026] a membrane bound protein on the surface of cells that is capableof binding one or more ligands.

[0027] Functional interaction—

[0028] an interaction between a receptor and ligand that results inactivation of cellular responses. These may include changes in membranepotential, secretion, action potential generation, activation ofenzymatic pathways and long term structural changes in cellulararchitecture or function.

[0029] Differentiated—

[0030] refers to the final structural and functional attributes of aparticular cell. The most mature state of a cell.

[0031] Cloned—

[0032] production or generation of a specific genetic sequence encodinga protein.

[0033] Recombinant—

[0034] a genetic construct in which a clone has been recombined in anovel genetic environment.

[0035] Recombinantly produced receptor—

[0036] a receptor protein produced by recombining its genetic sequence(clone) with other genes that induce the transcription and generation ofthe protein.

[0037] In vivo—

[0038] refers to preparations or methods performed in a living, intactanimal or organism, whether conscious or not.

[0039] In situ—

[0040] refers to methods performed on tissue that remains in its normalplace in the animal although the organism may be post mortem

[0041] In vitro—

[0042] refers to methods performed on tissues or cells that have beendissected free of an animal or are dissociated from the animal ororganism. The most common example is cell culture.

SUMMARY OF THE INVENTION

[0043] The present invention relates to methods and materials for theexpression and assaying of functional neuronal receptors in vivo. Moreparticularly, the present invention relates to methods and materials forthe expression and assaying of functional neuronal receptors, such asodor receptors, in neuronal cells, such as olfactory cells, in animals,such as rats. More particularly still, the present invention relates tothe use of recombinant viruses, such as recombinant adenoviruses,containing odorant receptor genes, to infect olfactory cells in ananimal and express functional cloned odorant receptors which can then beassayed for specific interaction with specific odorant molecules. Forexample, an embodiment of the present invention uses recombinantadenovirus (Adv) encoding an odorant receptor expression cassette alongwith a marker gene to infect rat nasal epithelia in vivo. This systemeffectively expresses a foreign odorant receptor gene that can beconveniently assayed for specific ligand activation by physiologicalmethods. Additionally, this invention identifies a set of ligands thatcan activate a particular receptor providing a method of pairing aparticular receptor of known amino acid sequence with a specific set ofodorant ligands.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1a illustrates the construct of the recombinant adenovirusAdexCAG-I7-IRES-GFP;

[0045]FIG. 1b shows the PCR amplification of E1 a and I7 fromAdexCAG-I7-IRES-GFP viral DNA;

[0046]FIG. 2a shows the side view of the medial surface of the rat nasalturbinates;

[0047]FIG. 2b shows the fluorescent micrograph of the same tissue as inFIG. 2a showing the heterogeneous expression of GFP which marked thelocation and the degree of virus infection;

[0048]FIG. 2c shows higher magnification view of the I7 virus infectedendoturbinate II′ in FIG. 2b;

[0049]FIG. 2d shows the Northern hybridization of total RNA fromuninfected olfactory epithelia, AdexCAG-I7-IRES-GFP virus infectedolfactory epithelia and rat brain with I7 probe;

[0050]FIG. 2e shows a cryosection of AdexCAG-I7-IRES-GFP infectedolfactory epithelium reacted with an antibody for GFP showing stainedolfactory neurons with characteristic morphology;

[0051]FIG. 3 shows a schematic diagram of electro-olfactogram recordingand odorant stimulation system;

[0052]FIG. 4 shows representative EOG recordings from an uninfected (a)and an AdexCAG-I7-IRES-GFP virus infected animal (b);

[0053]FIG. 5a shows the comparison of average EOG amplitudes inAdexCAG-I7-IRES-GFP virus infected (black bar) and uninfected animals(gray bar) to 14 odorants from the panel of 74;

[0054]FIG. 5b shows I7 virus infected animals have increased odorantresponse to heptaldehyde (C7), octyl aldehyde (C8), nonyl aldehyde (C9),and decyl aldehyde (C10), but not to hexaldehyde (C6) nor undecylicaldehyde (C11);

[0055]FIG. 5c shows a comparison of responses (EOG amplitude) in aninfected and uninfected animal to increasing concentrations of amylacetate and octyl aldehyde;

[0056]FIG. 5d shows the chemical structure of octyl aldehyde;

[0057]FIG. 6a shows freshly dissociated rat olfactory neurons (arrows)which can be easily identified by their morphology;

[0058]FIG. 6b shows the same field as in FIG. 6a under fluorescenceillumination, an olfactory neuron infected by AdexCAG-I7-IRES-GFP virusthat can be identified by expression of GFP; and

[0059]FIG. 6c shows a whole-cell patch clamp recording of a response to0.5 mM octyl aldehyde in an infected cell.

DETAILED DESCRIPTION OF INVENTION

[0060] The present invention provides methods and materials for theconstruction of expression vectors containing cloned neuronal receptors,such as putative odor receptors, and materials and methods for theintroduction of such vectors into neuronal cells (via for example,infection or transfection), such as olfactory cells, in vivo and theexpression of functional cloned receptors in such cells. Further, thepresent invention provides materials and methods for assaying forfunctional receptor binding activity of such cloned receptors,including, but not limited to applications such as identifying aparticular ligand for a particular receptor and/or quantitating andqualitating the binding activity of a particular receptor with aparticular ligand.

[0061] The invention also provides methods and materials for identifyingodorant ligands and for identifying odorant receptors. The inventionfurther provides methods and materials for, for example, developingfragrances, identifying appetite suppressant compounds, controllingappetite, controlling insect and other animal populations, enhancing ananimal's sense of smell, including to a particular odorant ligand ortype of ligand including, for example, detecting specific odors such asthe vapors emanating from cocaine, marijuana, heroin, hashish, angeldust, gasoline, decayed animal flesh, including human flesh, alcohol,gun powder explosives, plastic explosives, firearms, poisonous orharmful smoke, natural gas and so forth.

[0062] Methods and materials of the present invention for identifying adesired odorant ligand may comprise contacting neuronal cells, such asolfactory cells, containing a specific recombinant gene encoding andexpressing a specific odorant receptor, with a series of odorant ligandsto determine which ligands bind to the receptors present on the cells.Such methods may be performed in vivo and/or with cells infected in vivoand removed for in vitro assay, and/or with cells infected in vivo andremoved for in vitro assay, yet the subject cells remaining situated insitu as in normal olfactory tissue.

[0063] Methods and materials of the present invention for identifying adesired odorant receptor may comprise contacting a series of neuronalcells containing a specific recombinant gene encoding and expressing aspecific odorant receptor, with a known odorant ligand and determiningwhich odorant receptor binds with the odorant ligand.

[0064] An embodiment of the present invention provides a method ofdetecting an odor which comprises: a) identifying an odorant receptorwhich binds the desired odorant ligand and b) imbedding the receptor ina membrane such that when an odorant ligand binds to the receptor soidentified, a detectable signal is produced. Examples of such detectablesignals are membrane potential changes, electrophysiological methodsincluding, but not limited to, extracellular recording—measuring changesin membrane potential from multiple cells by use of an electrode placednear to, but outside of the cells; intracellular recording—measurementof membrane potential of a single cell by inserting a fine tippedelectrode into the cell interior; recording en passant, or suctionelectrode recording—measurement of electrical activity in a single cellby placing a blunt electrode tightly against the surface membrane of acell, but not penetrating the cell interior; whole cell patch clamprecording—measurement of membrane potential or ion current flow in asingle cell by means of an electrode which has access to the cellinterior, and single channel patch recording—measurement of the ioncurrents passing through one or a few ion channels in an isolated patchof membrane removed from a cell with a specialized electrode. In oneembodiment of the invention, the membrane used in this method iscellular, including a membrane of an olfactory cell or a syntheticmembrane.

[0065] The ligand tested for may be the vapors emanating from cocaine,marijuana, heroin, hashish, angel dust, gasoline, decayed human flesh,alcohol, gun powder explosives, plastic explosives or firearms. Inanother embodiment, the ligand tested for may be natural gas, apheromone, toxic fumes, noxious fumes or dangerous fumes.

[0066] In one embodiment of this method, the detectable signal is ameasurement of the change in the transepithelial potential (or membranepotential) across the cells' surface due to the interaction of ligandand receptor. The invention may further provide a method of quantifyingthe amount of an odorant ligand present in a sample, and/or the affinityof the receptor for the ligand, which comprises utilizing theabove-mentioned method for odor detection and then quantifying theamount of signal produced.

[0067] The invention further provides a method for developing fragranceswhich comprises identifying a desired odorant receptor by theabove-mentioned method, then contacting cells containing and expressingthe cloned odorant receptor in the above-described method with variousligands in order to determine which compounds bind the receptor and thecharacteristics of this binding.

[0068] The method can also provide a method for identifying an “odorantfingerprint” which comprises contacting a series of cells containing andexpressing known odor receptors with a desired sample, and determiningthe type and quantity of the odorant ligands present in the sample.

[0069] The invention also provides a method for identifying odorantligands which inhibit the activity of a desired odorant receptor (forexample a receptor agonist or antagonist) which comprises contacting thedesired odorant receptor with a series of compounds and determiningwhich compounds inhibit the odorant ligand-odorant receptor interaction.

[0070] The methods and materials of the invention can also be used in,for example, methods for identifying appetite suppressant compounds andusing same to suppress and/or control appetite; trapping odors of aspecific type; controlling pest populations by, for example, identifyingalarm odorant ligands and spraying same in areas in order to deter pests(such as insects, mice or rats) and/or using the inventive methods andmaterials to interfere with pest reproduction; as a method for promotingfertility or inhibiting fertility; and, for example, in creatinganimals, such as sniffing dogs, that are especially sensitive to certainodors, such as the vapors of drugs and explosives. Strategy forExpressing Odorant Receptors We have relied on the large number ofodorant receptors, and their approximately equal expression among thesix million neurons of the rat olfactory epithelium, to identify theaverage increase in response if one of these receptors could beoverexpressed in the epithelium. From existing data it is reasonable toassume that each of the approximately 1000 receptors is expressed inroughly 0.1% of the total cells, so that if expression of one particularreceptor could be induced in as few as 1-10% of the neurons theadditional response due to activation of that receptor by its particularligands would be relatively easy to detect.

[0071] The olfactory sensory epithelium lining the nasal cavity is acomparatively simple tissue consisting of only a single type of neuron,the olfactory receptor cell, as well as a glial cell type, thesustentacular cell. When activated by odorants the neurons depolarizeand generate action potentials. This activity can be measuredextracellularly as a transepithelial potential due to the summedactivity of many cells—a measurement known as the EOG orelectro-olfactogram (24). The amplitude of this voltage is determined byboth the size of the response in individual cells and the number ofcells responding. Electrical activity can also be measured byextracellular recording, intracellular recording, recording en passant,suction electrode recording, whole patch clamp recording, and singlechannel patch recording.

[0072] In order to induce olfactory neurons to express a particularreceptor we generated an adenovirus vector, AdexCAG-I7-IRES-GFP, thatcontained the gene for a particular odorant receptor (rat I7(3)), andinfected rat olfactory epithelia in vivo by direct application of abuffer containing the viral particles to the surface of the epithelium.Adenovirus, the carrier of the common cold, is a DNA virus that is knownto infect epithelia of the respiratory tract (20, 21). The E1 region ofthe viral genome is required for viral replication (see, FIG. 1); itsremoval renders the virus replication incompetent and provides space forthe insertion of foreign DNA. This recombinant virus can be produced inthe complementary HEK 293 cell line which contains the E1 adenovirusgenes (see Example 1). However, the viral particles produced are capableonly of a single infection and cannot replicate in other host cells.

[0073] From earlier work using an adenovirus containing the lacZ markergene, a strong, but heterogeneous, viral infection and proteinexpression in rat nasal epithelium has been observed(25, 26). Tofacilitate electrode placement and maximize the electrical signal, or toeasily locate individual transfected cells, it is useful to know theparticular regions on the epithelium in which the virally deliveredreceptor is expressed in any individual animal. Therefore, a visible orother cellular marker can be expressed, for example, the gene for thephysiological marker green fluorescent protein (GFP) was inserted in theexpression cassette of Example 1 (see, FIG. 1). Because of concerns thata receptor-GFP fusion protein might alter protein expression orfunction, we utilized an IRES (internal ribosomal entry site) insert toproduce a bicistronic message (27, this reference is hereby incorporatedby reference in its entirety) that would result in the expression ofodorant receptor and GFP as separate proteins in the same cells. Weselected the rat I7 odorant receptor (3) for expression, but anyreceptor should work in this system.

[0074] The following Examples are intended to illustrate the embodimentsof the present invention and are not in any way intended to limit thescope of the invention in any manner.

EXAMPLE 1 Adenoviral Vector Construction

[0075]FIG. 1a illustrates the construct of the recombinant adenovirusAdexCAG-I7-IRES-GFP. The replication-defective adenovirus expressionvector Adex consists of the human adenovirus type 5 (Ad5) genome lackingthe E1a, E1b and E3 regions. AdexCAG-I7-IRES-GFP has a bicistronicexpression unit including the CAG promoter (CAG), composed of thecytomegalovirus enhancer plus the chicken beta-actin promoter, odorantreceptor I7 (OR-I7) coding sequence, internal ribosomal entry site(IRES), the fragment for the S65T mutant of green fluorescent protein(GFP), and rabbit beta-globin polyadenylation signal (GpA). The CAGpromoter drives transcription of the bicistronic message I7-IRES-GFPproducing I7 odorant receptor and GFP as separate proteins. Any othersuitable transfection vector can be used, as will be appreciated by oneskilled in the art.

[0076] The entire odorant receptor coding sequence was amplified bypolymerase chain reaction (PCR) (see, FIG. 1b), using the pfu DNApolymerase (Stratagene) from the I7 clone plasmid provided by Dr. L.Buck with the upstream primer: SEQ ID NO. 15′ CCCTCGAGTATGGAGCGAAGGAACCAC 3′

[0077] and the downstream primer: SEQ ID NO. 25′ GCTCTAGACTAACCAATTTTGCTGCCT 3′

[0078] The 0.6 kb internal ribosomal entry site (IRES) fragment (27) wascut with EcoRI and BamHI from plasmid p1162 provided by Dr. ThomasLufkin. The fragments of I7, IRES and the S65T mutant of greenfluorescent protein (GFP) were first conjugated in the polycloning sitesof the expression vector pCA4 (Microbix, Ontario, Canada) and tested byNorthern blot with an I7 probe for transcription of mRNA, and greenfluorescence for IRES-driven GFP expression in human embryonic kidney(HEK) 293 cells (ATCC, CRL-1573). The I7-IRES-GFP sequence was thensubcloned into the Swal site of the cosmid vector pAdexlpCAw (38, 39).The pAdexlpCAw cosmid was created from the human adenovirus type 5genome from which the E1a, E1b, and E3 regions were deleted and replacedwith an expression unit containing the CAG promoter (composed of thecytomegalovirus enhancer plus the chicken beta-actin promoter (40)), aSwal site, and the rabbit beta-globin polyadenylation signal. The I7sequence in the cosmid vector pAdexI7-IRES-GFP was confirmed bysequencing. The cosmid vector pAdexI7-IRES-GFP and the EcoT22I digestedDNA-terminal protein complex (DNA-TPC) (41) of Ad5-dlX which is a humantype 5 adenovirus lacking the E3 region were co-transfected into HEK 293cells by calcium phosphate precipitation. The recombinant adenovirusAdexCAG-I7-IRES-GFP was then generated by homologous recombination inthe HEK 293 cells. The DNA-TPC method has been described in detail inrefs. 39 and 41 (these references are hereby incorporated by referencein their entirety). BamHI and XbaI digestion of the genomic DNA ofAdexI7-IRES-GFP produced the appropriate band pattern, and positive PCRamplification of I7 also verified the construct (see, FIG. 1b). Sincerecombinant viruses do not include the E1a genes, PCR amplification ofthe E1a region was performed with the primers: SEQ ID NO 3:5′ ATTACCGAAGAAATGGCCGC 3′ and SEQ ID NO 4: 5′ CCCATTTAACACACGCCATGCA 3′

[0079] as a control for contamination by wild type adenovirus (Ad5-dlX).

[0080]FIG. 1b shows the PCR amplification of E1a (lane 1) and I7 (lane2) from AdexCAG-I7-IRES-GFP viral DNA. The completely negativeamplification for El a confirmed the absence of the wild type adenoviruscontamination. The positive amplification for I7 (Lane 2) andrestriction enzyme digestions were used to verify the viral construct.Lanes 3 and 4 are E1a and I7 amplified from plasmids containing El asequence and I7 sequence respectively as the positive amplificationcontrols. Negative PCR amplification of the E1a gene was observed inevery stock of recombinant adenovirus. The recombinant adenovirus waspropagated in HEK 293 cells and purified by cesium gradientcentrifugation (42). The viral titer was determined by plaque formingassay on HEK 293 cells.

[0081] Techniques for performing such as the above-mentioned and someother illustrative examples herein presented, are well known to, andunderstood by, those skilled in the art (see, for example, PCTApplication No. PCT/US92/02741 (WO 92/17585) which is herebyincorporated by reference in its entirety).

EXAMPLE 2 Infection and Expression in Nasal Epithelia

[0082] Rats (Sprague-Dawley) of various ages and both sexes were usedfor these experiments. Under anesthesia (ketamine, 90 mg/kg andxylazine, 10 mg/kg, i.p.), 30 μl of rat Ringer solution (in mM, 135,NaCl; 5, I(Cl; 1, CaCl₂; 4, MgCl₂; 10, HEPES; pH 7.4) containing theAdexCAG-I7-IRES-GFP at a titer of 3×10⁹ pfu/ml, and 0.3% fast green dyewas slowly injected through the nostril into the right side of the nasalcavity with a short length of thin plastic tubing. The solution wasallowed to remain in the nasal cavity. After recovery, the animals weremaintained at room temperature with no other treatment until sacrificed.

[0083] Approximately 30 μl of buffer containing the purified recombinantadenovirus, AdexCAG-I7-IRES-GFP, at a titer of 3×10⁹ pfu/ml wasirrigated into the nasal cavity of anesthetized rats of varying ages andsex. The animals were sacrificed 3-8 days later and the nasal cavityopened, exposing the medial surface of nasal turbinates (see, FIG. 2a).FIG. 2a shows the side view of the medial surface of the rat nasalturbinates. The turbinafes are labeled with Roman numerals. Dorsal isup, anterior to the left. This animal was infected byAdexCAG-I7-IRES-GFP (scale bar=3 mm).

[0084] The olfactory turbinates were dissected out and fixed with 4%paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) for 2 hoursand cryoprotected in 20% sucrose. 15 μm cryostat sections were cut andincubated with the polyclonal antibody for GFP (CLONTECH Laboratories).Specific staining was then visualized by using Vectastain Elite ABC kit(Vector Lab).

[0085] Under fluorescent illumination the GFP could be visualized easily(see, FIG. 2b and 2 c), clearly marking the pattern of viral infectionand protein expression. FIG. 2b shows the fluorescent micrograph of thesame tissue as in FIG. 2a showing the heterogeneous expression of GFPwhich marked the location and the degree of virus infection (scale bar=3mm). FIG. 2c shows higher magnification view of the I7 virus infectedendoturbinate II′ in FIG. 2b. In regions of high fluorescence weestimate that infection rates were near 10% of neurons (scale bar=1 mm).

[0086] In some regions of the epithelia as many as 20% of the neuronswere infected, while in others there was virtually no sign of infection.Overall about 1-2% of the sensory neurons were infected and expressedthe GFP gene product. The highest infection rates were typically foundin the second and third turbinates, usually near the edges (see, FIG.2c).

EXAMPLE 3 Detection of Bicistronic mRNA Expression of The I7 Receptorand GFP

[0087] Expression of the bicistronic mRNA for the I7 receptor and GFPwas verified by Northern blot of the infected epithelia. Northern blotdetection of I7 mRNA was performed using a standard procedure (43).Total RNAs were extracted from tissues with TRIzol reagent (GibcoBRL).20 μg of total RNA was loaded on each lane of the gel. The I7 probe wassynthesized by PCR with primers that covered the entire I7 codingsequence, and labeled with Digoxigenin (DIG-11-dUTP, BoehringerMannheim) according to the manufacturer's protocol. After hybridizationthe probe was detected with the DIG Nucleic Acid Detection Kit(Boehringer Mannheim).

[0088] Using a probe that covered the entire sequence of the I7 gene wedetected a single band of about 3 kb in infected, but not in uninfectedepithelia where it is presumably below the level of detection (see, FIG.2d). FIG. 2d shows the Northern hybridization of total RNA fromuninfected olfactory epithelia (uninfected OE), AdexCAG-I7-IRES-GFPvirus infected olfactory epithelia (infected OE) and rat brain with I7probe. 20 micrograms of total RNA from each tissue were loaded in eachlane. Examination of 28S and 18S rRNA confirmed the integrity of theRNA.

[0089] Although this does not provide a quantitative measure of theextent of mRNA expression, it does provide clear evidence thatexpression of the I7 receptor message is much higher in infected versusuninfected tissue. For our purposes the precise rate of mRNAtranscription may not be critical since the number of cells makingcloned receptor has a greater effect on the EOG than the amount ofreceptor being made by any single neuron. This is because the gainamplification of the second messenger system in olfactory neuronsassures that even the activation of a few receptors by ligand willproduce a significant sensory current in individual cells. This isevident in recordings from single cells where odorant induced currentsin virus infected cells are comparable to those in normal cells (seebelow). In the absence of antibodies specific for the I7 odorantreceptor, we utilized GFP antibodies to further verify that, as with thelacZ adenovirus, the infection rate was much greater in the olfactorysensory neurons than in sustentacular cells (see, FIG. 2e). FIG. 2eshows a cryosection (15 μm) of AdexCAG-I7-IRES-GFP infected olfactoryepithelium reacted with an antibody for GFP showing stained olfactoryneurons with characteristic morphology, including soma (arrowhead) andsingle dendrite (arrow), and position within the olfactory epithelium.ML, mucous layer; OE, olfactory epithelium; BL, basal lamina (scalebar=50 μm). Thus areas of high GFP fluorescence signal the positiveinfection of sensory neurons.

EXAMPLE 4 Electro-olfactogram (EOG) Recording

[0090] The animal was overdosed with anesthetics (Ketamine and xylazine)and decapitated. The head was cut open sagittally and the septum wasremoved to expose the medial surface of the olfactory turbinates (44this reference is hereby incorporated by reference in its entirety). Theright half of the head was mounted in a wax dish filled with rat Ringer.The medial surface of turbinates was face up and exposed to the air. Acontinuous stream of humidified clean air was gently blown on theturbinates through tubing to prevent tissue from drying. The opening ofthe tubing was 8 mm in diameter and placed approximately 10 mm from theturbinate surface.

[0091] Odorant solutions were prepared by diluting a 0.5 M stock in DMSOwith water. All odorant chemicals were purchased from Aldrich, exceptLyral and Lilial which were the kind gift of IFF and Harmon & ReimerInc. Three milliliters of the odorant solution were placed in a 10 mlglass test tube and capped with a silicon stopper. The concentration ofvolatile odorant in the 7 cc airspace was allowed to equilibrate formore than 1 hour. All solutions were used within 8 hours. Two 18 gaugeneedles provided the input and output ports for the odorant containingvapor above the solution. For stimulation a 100 ms pulse of the odorantvapor at 9 psi was injected into the continuous stream of humidifiedair. The pulse was controlled by a Picospritzer solenoid controlledvalve (General Valve). Amyl acetate was used as the reference odorant tocontrol for the variability in responses between animals and during longrecording sessions, and was delivered on every sixth trial of odorantstimulation. Three tubes of amyl acetate at 10⁻³ M liquid concentrationwere used alternately in each experiment. The odorant stimulus pathwaywas cleaned by air between each stimulus presentation. The minimuminterval between two adjacent stimuli was 1 minute.

[0092] The EOG recording electrode was an Ag/AgCl wire in a capillaryglass pipette filled with rat Ringer solution containing 0.6% agarose.The electrode resistance was between 0.5 and 1 MΩ. The recording pipettewas placed on the surface of the olfactory epithelium and connected to adifferential amplifier (DP-301, Warner Instrument). Placement of theelectrode was determined by visualizing GFP fluorescence with a modifiedstereomicroscope (Kramer Scientific). The EOG potential was observed ona chart recorder, recorded with a DAT tape recorder, and latertransferred to computer. For most experiments two electrodes and twoamplifiers were used to record EOGs from two different sites ofepithelium simultaneously. All experiments were performed at roomtemperature (22-25° C.).

EXAMPLE 5 Odorant Responses in Infected Epithelia

[0093] One difficulty in determining the ligand specificity of odorantreceptors is the enormous stimulus repertoire to be tested. Well over2,000 odorous chemicals are cataloged, including substances fromvirtually all classes of organic molecules. We developed a panel of 74odorants including aromatic and short chain aliphatic hydrocarbons withvarious functional groups, including aldehydes, alcohols, alkanes,esters, acids, ketones, esthers and amines. For a complete list of theodorants tested, see Table 1. This list is by no means to be construedas a comprehensive list of candidate ligands but is provided merely forillustrative purposes. TABLE 1 List of Odorants Tested AROMATICSAldehydes Alcohols Ketones Ethers para- Cinnamyl Aceto AnisoleAnisaldehyde alcohol phenone (−) Carvone Eugenol 2-Decalone CineoleCinnamaldehyde Guaiacol 2-Methylanisole Salicylaldehyde 4-MethylanisoleLilial Isoeugenol Methyl eugenol Esters Hydrocarbons HeterocyclesCinnamylformate ortho(2)- 2-Isobutyl-3- Ethyltoluene methoxypyrazineGeranyl acetate meta(3)- Ethyltoluene Isoamyl salicylate ortho(1,2)-Diethylbenzene Linalyl formate Limonene ALIPHATICS Acids AldehydesAlcohols Octanoic acid Propion aldehyde n-Propyl alcohol n-PelargonicNonanoic acid Isobutyr-aldehyde n-Butyl alcohol Propionic acid n-Hexylaldehyde n-Pentyl alcohol n-Valeric acid n-Heptaldehyde n-Hexyl alcoholn-Octyl aldehyde n-Heptyl alcohol Alkanes trans-2-Octenal n-Octylalcohol n-Octane 2-octynal n-Nonyl alcohol n-Nonane n-Nonyl aldehyden-Decyl alcohol n-Decane n-Decyl aldehyde 2-Ethylfenchol Dodecylaldehyde Geraniol Amines Undecylic aldehyde B-Citronellol Isopentylaminetrnas-2-Tridecanal Linalool Phenethylamine Citral n-ValeraldehydeKetones Esters Lyral 2,3-Butanedione Amyl acetate 1-Fenchone Ethylbutyrate Ethers 2-Nonanone Ethyl hexanoate Citral diethyl acetal Isoamylacetate Citral dimethylacetal Octyl butyrate Octyl isovalerate OtherHeptyl cyanide 1,1,3,3- Tetramethylbutyl isocyanide isocyanide

[0094] Odorants were prepared and applied in the following manner. Threemilliliters of each odorant solution at a particular concentration wereplaced in a 10 ml test tube stoppered with a silicone cap, and left forone hour to allow the concentration of volatile odorant in the 7 ccairspace above the solution to equilibrate. Most of the odorants wereprepared at a liquid concentration of 10⁻² M to 10⁻³ M depending ontheir relative volatilities. Concentrations of stimulus produced at theolfactory epithelium could not be known, but the system reliablydelivered the same amount of stimulus on each trial as there was littlevariability between responses to successive pulses of the same odorant.The absolute odorant concentration at the tissue was not critical sinceresponses to all odorants were compared in different animals and in eachanimal the responses were normalized to that animal's response to amylacetate, the standard odorant.

[0095] The stimulus delivery and EOG recording methods are picturedschematically in FIG. 3. FIG. 3 shows a schematic diagram ofelectro-olfactogram recording and odorant stimulation system, asdescribed in the text. The inset shows 4 responses to odorants thatrepresent the range of response types observed. OB is the olfactorybulb. The dashed line running diagonally across the epithelium indicatesthe border between sensory and non-sensory respiratory epithelia.

[0096] Pulses of clean air lasting 100 msec at 9 psi were used to injecta bolus of the odorant containing vapor into a continuous stream ofhumidified, clean air directed at the exposed sensory epithelium. Underthese conditions the tissue remained viable for up to 3 hours (althoughall recordings presented here were completed in under two hours), and itwas possible to run twice through a panel of 30-50 odorants with eachtrial separated by at least 1 minute. There was no systematic differencein viability between animals of different ages or sexes, nor betweeninfected and non-infected control animals.

[0097] The EOG recording method, pictured schematically in FIG. 3,consists of a single glass capillary electrode connected to adifferential amplifier. This electrode, placed on the epithelialsurface, records transepithelial potentials that arise as a result ofthe depolarizing ionic currents of multiple olfactory neurons during astimulus induced response. The EOG is recorded as a negative potentialthat rises rapidly to a spike-like peak which varies in maximumamplitude from less than a millivolt to as much as 15 mV, and thendecays with a variable time course ranging from 1 to 5 seconds.Representative recordings of responses to several odorants from a normalrat olfactory epithelium are shown in the inset to FIG. 3. There wassignificant variability in the responses between animals to the variousodorants, and, during long recording sessions lasting up to 2 hours,there was also variation in the responses over time. These are wellknown attributes of the EOG recording method, and in order to controlfor this variability we utilized a standard odorant, amyl acetate, towhich all other odorant responses were normalized. To control for thetemporal variability the amyl acetate standard was delivered every sixthtrial and intervening responses were normalized to the average of thepreceding and following amyl acetate responses.

[0098] Since the contribution of cells near to the electrode is greaterthan that from distant cells the placement of the electrode is critical.With a modified fluorescent stereomicroscope (Kramer Scientific, NY) wewere able to visualize the GFP distribution and place our EOG electrodein a region of the olfactory epithelium that showed high levels offluorescent (i.e. infected) cells. All infected animals were able torespond to all of the 74 odorants in the test panel, however responsesin virally infected animals were on average 30% smaller in amplitude.Additional markers include, but are not limited to, lac-z—the gene forthe bacterial enzyme beta-galactosidase, cells expressing this proteinwill turn a dark blue color upon reaction with the substrate X-gal;epitope tags—specialized short genetic sequences which insert a specificsequence of amino acids in a region of a protein so that the protein canbe identified by antibodies directed against the “epitope tag” sequence;and fluorescent markers—including GFP and other substrates that can becaused to fluoresce when exposed to specific wavelengths of light.

[0099] Responses to 8 representative odorants from the panel of 74odorants screened are shown in FIG. 4. FIG. 4 shows representative EOGrecordings from an uninfected (a) and an AdexCAG-I7-IRES-GFP virusinfected animal (b). All EOGs are responses to odorants at a solutionconcentration of 10⁻³ M and are normalized to the reference odorant.

[0100] All responses are normalized to the standard (amyl acetate)odorant response which is given the value of unity. That is, all otherresponses are shown as the ratio of the response amplitude to theamplitude of the amyl acetate response in that animal. For 7 of the 8odorants shown (plus the amyl acetate standard), as for 62 of the 65other odorants in the panel, there was no significant change inresponsiveness between infected and uninfected animals. However, for oneodorant, octyl aldehyde or octanal, an eight carbon straight chainaliphatic aldehyde, the response is dramatically greater, both inamplitude and time course, in the infected animal. Octyl aldehyde isvariously described as having a citrus, soapy and/or fatty odor quality(for the chemical structure, see FIG. 5d).

[0101] The dramatic response for octyl aldehyde in the infected animalcompared to the uninfected animal can be seen even more clearly in thetraces of FIG. 4c which are the result of subtracting the responses inthe normal epithelium from those in the infected animal which brings outthe much increased response to octyl aldehyde in the AdexCAG-I7-IRES-GFPvirus infected animal. The slight positive direction of some of thesubtracted traces is primarily due to differences in the time course ofthe responses in the different animals. The significant increase inamplitude and time course indicates that more individual olfactoryneurons were responsive to octyl aldehyde in the infected versusuninfected tissue.

[0102] The average response amplitudes for 14 of the odorants from thepanel of 74 are compared graphically in FIG. 5a. FIG. 5a shows thecomparison of average EOG amplitudes in AdexCAG-I7-IRES-GFP virusinfected (black bar) and uninfected animals (gray bar) to 14 odorantsfrom the panel of 74. All responses were normalized to the referenceodorant, amyl acetate. The responses in uninfected animals are given thevalue of unity, and the responses in infected animals are scaledaccordingly. As a further control, the response to octyl aldehyde wascompared to that in animals infected with an adenovirus carrying onlyGFP (open bar). All averages are from 6 to 27 trials, error bars are thestandard deviation (S.D.).

[0103] Octyl aldehyde responses are, on average, 1.7 times greater ininfected vs. uninfected animals, while all other odorants are nearcontrol levels. The responses to octyl aldehyde are also compared to acontrol in which animals were infected with a similar amount of anadenovirus containing only the GFP gene driven by the same CAG promoter(23). Expression of GFP in these animals was comparable to that seen inanimals infected with the AdexCAG-I7-IRES-GFP virus, but the response tooctyl aldehyde was not different from uninfected animals. Thus, neitherviral infection nor GFP alone was sufficient to generate the increase inresponsivity to octyl aldehyde.

[0104] Other related odorants were tested to see if they were recognizedby the I7 receptor. FIG. 5b shows I7 virus infected animals haveincreased odorant response to heptaldehyde (C7), octyl aldehyde (C8),nonyl aldehyde (C9), and decyl aldehyde (C10), but not to hexaldehyde(C6) nor undecylic aldehyde (C11). The bars are the result ofsubtraction that shows the fold increase of the average responses (n=4to 27) to these odorants in infected animals vs. uninfected animals.Other 8 carbon aliphatic compounds with different functional groups, andat least one 8 carbon unsaturated aliphatic aldehyde, trans-2-octenal,failed to elicit responses larger than in normal animals. Thus theresponse profile of the I7 receptor, at least within the scope of the74-odorant panel screened here, is relatively specific for saturatedaliphatic aldehydes with straight chains between 7 and 10 carbons inlength.

[0105] At the comparatively high concentrations used in theseexperiments the response to octyl aldehyde was on average 1.7 timesgreater in infected versus control animals. In fact, these highconcentrations, because they are near the saturation point for the octylaldehyde response, mask the full effect of the virally induced odorantreceptor expression. FIG. 5c shows a comparison of responses (EOGamplitude) in an infected and uninfected animal to increasingconcentrations of amyl acetate (AA, triangles) and octyl aldehyde (OA,squares). Heavy lines (and filled symbols) are from the infected animal,light lines (open symbols) are from the uninfected control animal. Thex-axis is the log of the molar concentration of odorant solutions.

[0106] As can be seen in the dose-response relations of FIG. 5c,comparison of the octyl aldehyde response to the amyl acetate at variousconcentrations revealed up to a 7-fold difference in response magnitudein infected versus control epithelia. For example, at an odorantconcentration (in solution) of 5×10⁻⁵ M, the response ratio of octylaldehyde to amyl acetate in infected animals was 0.6:4.0 mV, 7 timeslarger; at the same concentration in normal animals the responses werenearly equal and the ratio was 1.

[0107] The EOG response is a function of both the number of respondingcells and the response amplitude in individual cells. The relativecontribution of each of these factors is not known, although as notedabove, the high gain of the signal transduction cascade in the olfactoryneuron makes it likely that the difference in EOG amplitudes betweencontrol and infected animals in these experiments is due primarily to anincreased number of cells expressing the octanal (octyl aldehyde)sensitive receptor. In either case, the more cells near the electrodethat are responding to a stimulus, the larger the response is likely tobe. In particular a larger population of cells should increase both theamplitude of the response and the time course, a result that we haveobserved here. Moving the EOG electrode from areas of high fluorescenceto regions with little or no GFP expression significantly reduced therelative amplitude of the octyl aldehyde response, but did not affectthe responses to other odorants. Similarly in animals with lowerinfection rates, as judged by the extent of GFP induced fluorescence,responses to octyl aldehyde were comparably smaller.

EXAMPLE 6 Responses in Single Olfactory Neurons

[0108] Although the EOG is a simple and convenient method forefficiently screening for sensitivity to a large number of odorants,more detailed data regarding the odorant response can be obtained byrecording from single olfactory neurons. Because the infected cellsexpressed GFP, they could be located by their fluorescence after thetissue was dissociated (see, FIGS. 6a, and 6 b). FIG. 6a shows freshlydissociated rat olfactory neurons (arrows) which can be easilyidentified by their morphology, while FIG. 6b shows the same field as inFIG. 6a under fluorescence illumination. An olfactory neuron infected byAdexCAG-I7-IRES-GFP virus can be identified by expression of GFP (scalebar=20 μm).

[0109] Whole-cell patch clamp recording was performed on isolated cellswith detectable fluorescence (FITC filter). Cell dissociation techniquesfor olfactory epithelium are described in detail elsewhere (45 thisreference is hereby incorporated by reference in its entirety); forthese experiments we included only papain treatment and mechanicaldisruption. Recordings were made with the Axopatch 1D amplifier.Whole-cell pipettes had a resistance of 5-10 MΩ and contained 135 mMCsCl; 1 mM CaCl₂; 1 mM MgCl₂; 10 mM EGTA; 10 mm HEPES; 4 mM, ATP; 0.3 mMGTP; pH 7.4. Data were acquired with the PULSE software (HEKA). Odorantswere applied by a constant stream perfusion device (SF77, WarnerInstruments) or by pressure pulse. The extracellular solution was therat Ringer described above. All cells were tested to confirm that theyhad the normal voltage gated currents before investigating their odorantresponses.

[0110] Whole-cell patch clamp recordings from isolated GFP positiveneurons revealed an octanal induced current in 5 of 5 cells (see, FIG.6c). FIG. 6c shows a whole-cell patch clamp recording of a response to0.5 mM octyl aldehyde in an infected cell. The holding potential was −60mV. The currents varied from 60-250 pA in response to a 50 msec pulse ofoctanal at approximately 10−4 M, values that are within the rangepreviously reported for rat olfactory neurons (28). They displayed thetypical time course of odorant induced currents, including a 100-250msec latency, a slow rise and exponential decay. The odorant inducedcurrent reversed around 0 mV. These data indicate that it is possible torecord odorant induced responses due to virally transferred receptorswith the resolution available in single cells.

[0111] The functional expression of a cloned odorant receptor provides acritical tool for developing an olfactory pharmacology in which odorantligands can be correlated with specific receptors. Thus, defining thereceptive field of an olfactory sensory neuron is a critical first stepin understanding how olfactory perception is achieved by higherprocessing in the central nervous system. Additionally, because theodorant receptors, with nearly 1000 different receptor genes, make upthe largest sub-family of G-protein coupled receptors (GPCRs), apharmacology of odorant receptors could enable us to better appreciatethe relation between gene sequence and binding specificity in thisimportant class of membrane receptors.

EXAMPLE 7 Implications for Olfactory Stimulus Coding

[0112] The success of the particular strategy employed here also hasseveral implications for the cell biology of olfactory neurons. For one,it demonstrates that olfactory neurons can express more than one odorantreceptor. That they probably do not normally do this (16) appears thento result from regulation at the transcriptional level, and not fromMRNA processing or translational control mechanisms. That is, thereappears to be no mechanism to prevent the translation, expression andproper processing of additional odorant receptors in a sensory neurononce the mRNA is generated. On the other hand, since these receptorshave not been successfully expressed in other heterologous expressionsystems, it does appear likely that olfactory neurons possess somecellular machinery specifically involved in the targeting and insertionof receptor proteins into the membrane. This should not be surprisingfrom the physiological determination that odorant sensitivity is largelylimited to the cilia (29, 30), suggesting that perhaps the samemolecular processes are also at work concentrating transduction enzymesin the ciliary compartment. We have used receptor clones that cover onlythe coding sequence of the receptor molecule, indicating thatuntranslated signal sequences are not essential.

[0113] It also appears that the odorant receptors may require a specificG-protein for effective coupling and signal transmission. G_(olf) is aG_(s) type of G-protein, but its expression is restricted almostentirely to olfactory neurons (31). One of the characteristics of thefamily of odorant receptors that distinguishes them from other GPCRs isa particularly short third intracellular loop between transmembranedomains 5 and 6 (3). This loop has been implicated in receptor G-proteininteractions in other GPCRs (32) and in the odorant receptors is only 17residues long, compared to 25-40 in other members of the superfamily(33). One earlier attempt to express odorant receptors (in sf9 cells)resulted in very small responses that saturated at low levels of secondmessenger production, indicating a possibly weak coupling betweenreceptor and endogenously available G-proteins (34). In the nativeolfactory neurons, as used in our strategy, a foreign odorant receptorappears to have no difficulty coupling to the endogenous pathway, asdemonstrated by the responses in single cells that are well within thenormal size range for physiological odorant responses (28).

[0114] The panel of odorants used to screen infected epithelia (see,Table 1) was drawn from an extensive physiological and psychophysicalliterature on odorant sensitivities. The odorants chosen were intendedto represent a large range of odorant qualities and chemical types. Inthe only other recent study in which such an extensive series ofodorants were utilized, responses to odorants applied to intactolfactory epithelium were recorded in mitral cells of the rabbitolfactory bulb (35). The mitral cell is the second order neuron ontowhich olfactory sensory neurons synapse in the region of neuropil knownas glomeruli. Mitral cells, each with dominant input from a single typeof olfactory receptor neuron, were found to have response profiles notunlike those observed here for the I7 receptor. That is, one particularodorant was usually the most effective at stimulating the mitral cell,but odorants with related structures were also excitatory, although to alesser degree. One important difference was that for mitral cells thecarbon chain length appeared to be more critical than the functionalgroup in determining stimulus efficacy.

[0115] We found that 7, 8, 9 and 10 carbon saturated aldehydes wereligands for this receptor. Aldehydes with chains of less than 7 or morethan 10 carbons were not ligands. Additionally other 8 carbon aliphaticswith different functional groups were not ligands. This suggests thatboth the functional group and the carbon chain length are criticaldeterminants of the odorant epitope. The only other odorant receptor forwhich a distinct ligand is known is the ODR-10 receptor in C. eleganswhich displays strong specificity for the chemical attractant diacetyl(36). This receptor appears more narrowly tuned than the rat receptortested here which may reflect the very different demands placed on thenematode and mammalian olfactory systems as regards the size of thestimulus repertoire from which ligands must be discriminated.

[0116] The nearly 1000 separate genes encoding receptors in theolfactory system provide an opportunity to explore structure-functionrelations in the GPCR superfamily. Genes of the odorant receptorsubfamily show a hypervariable region corresponding to the secondthrough fifth transmembrane domains (3), the presumed ligand bindingsite in GPCRs (32). However, in some cases odorant receptors of the samesubfamily differ from each other by only a few residues in this region(37). These genetically closely related receptors can now be tested todetermine if small sequence differences result in significant changes inligand sensitivity. With this tool it will be possible to identify keyamino acid residues which could then be mutated in recombinant receptorconstructs. Such a program could lead to a detailed, experimentallytestable understanding of the relation between gene sequence and ligandbinding specificity in membrane bound receptors.

[0117] The foregoing disclosure and description of the invention areillustrative and explanatory thereof, and various changes in the detailsof the illustrated apparatus and construction and method of operationmay be made without departing from the spirit of the invention.

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What is claimed is:
 1. A method for screening for cellular receptorshaving a particular receptor ligand specificity comprising the steps of:expressing at least one recombinantly produced cellular receptor in acell located amongst intact tissue; contacting said recombinantlyproduced cellular receptor expressing cell with at least one candidateligand; and assaying for functional interaction between saidrecombinantly produced cellular receptor and said candidate ligand. 2.The method of claim 1, wherein said recombinantly produced cellularreceptor is expressed in said cell in vivo.
 3. The method of claim 1,wherein said recombinantly produced cellular receptor is expressed insaid cell in situ.
 4. The method of claim 1, wherein said cell is adifferentiated cell.
 5. The method of claim 1, wherein said cell is anolfactory sensory neuron.
 6. The method of claim 1, wherein said cell isin a mammal's nasal epithelium.
 7. The method of claim 1, wherein thecellular receptor is selected from the group consisting of thesuperfamily of G protein coupled receptors.
 8. The method of claim 1,wherein the cellular receptor is a neuronal receptor.
 9. The method ofclaim 1, wherein the cellular receptor is an odorant receptor.
 10. Themethod of claim 9, wherein the odorant receptor is odorant receptor I7.11. The method of claim 1, wherein the contact of said recombinantlyproduced cellular receptor expressing cell with at least one candidateligand is via airborne administration of said candidate ligand.
 12. Themethod of claim 1, wherein said candidate ligand is selected from thegroup consisting of cellular receptor agonist molecules and cellularreceptor antagonist molecules.
 13. The method of claim 1, wherein thecandidate ligand is an odorant molecule.
 14. The method of claim 1,wherein the candidate ligand is selected from the group consisting ofthe ligands identified in Table
 1. 15. The method of claim 1, whereinthe recombinantly produced cellular receptor is odorant receptor I7 andthe candidate ligand is selected from the group consisting of heptylaldehyde, octyl aldehyde, nonyl aldehyde, and decyl aldehyde.
 16. Themethod of claim 1, wherein the recombinantly produced cellular receptoris produced from a recombinant construct comprising a cloned cellularreceptor and an adenovirus.
 17. The method of claim 16, wherein theadenovirus is human adenovirus type 5 and the cellular receptor isodorant receptor I7.
 18. The method of claim 1, wherein said assay forfunctional interaction comprises measuring the change in membranepotential of one or more of said cells in response to said candidateligand.
 19. The method of claim 1, wherein the assay for functionalinteraction between said recombinantly produced cellular receptor andsaid candidate ligand comprises the step of: measuring the electricalactivity of one or more of said cells containing said recombinantlyproduced cellular receptor in response to said candidate ligand.
 20. Themethod of claim 19, wherein said electrical activity is measured bymeans of extracellular recording, intracellular recording, recording enpassant, whole-cell recording using patch clamp, and single channelrecording.
 21. The method of claim 20, wherein said cell is a singlecell.
 22. The method of claim 1, further wherein: said intact tissuecontaining said cell is isolated in vitro prior to contact with saidcandidate ligand.
 23. The method of claim 1, further wherein: at leastone recombinantly produced marker is expressed with the expression ofsaid recombinantly produced cellular receptor in said cell, said markerthereby identifying cells containing said recombinant receptor.
 24. Themethod of claim 23, wherein said marker is selected from the groupconsisting of fluorescent markers and histochemical markers.
 25. Acomposition of matter comprising: a receptor-ligand complex ofrecombinantly produced odorant receptor I7 and a ligand selected fromthe group consisting of straight chain aldehydes having from 7 to 10carbons in length.
 26. The composition of claim 25, wherein the ligandis octyl aldehyde.
 27. The composition of claim 25, further comprising aneuronal cell in which said recombinantly produced odor receptor I7 hasbeen produced and expressed.
 28. The composition of claim 27, whereinthe neuronal cell is a nasal epithelial cell.
 29. A method for screeningfor neuronal receptors having a particular receptor ligand specificitycomprising the steps of: expressing at least one recombinantly producedneuronal receptor in a neuronal cell located amongst intact tissue;isolating intact tissue expressing said recombinantly produced neuronalreceptor; contacting said recombinantly produced neuronal receptorexpressing cell with at least one candidate ligand; and assaying forfunctional interaction between said recombinantly produced neuronalreceptor and said candidate ligand.
 30. The method of claim 29, whereinsaid neuronal receptor is an odorant receptor, said neuronal cell is anolfactory cell, and said ligand is an odorant ligand.
 31. A method forscreening for ligands having a particular cellular receptor specificitycomprising the steps of: expressing at least one recombinantly producedcellular receptor in a cell located amongst intact tissue; contactingsaid recombinantly produced cellular receptor expressing cell with atleast one candidate ligand; and assaying for functional interactionbetween said recombinantly produced cellular receptor and said candidateligand.